Liposome is a lipid vesicle composed of a phospholipid bilayer and has been attempted to find its application in various fields. Attention has been paid, in particular, to the application to drug carrier and sensors for diagnosis and detection, where large problems have been encountered in providing the liposome with a specific function by fixing a special functional substance onto or into the liposome and in maintaining the concentration of such liposome cells in the blood.
Heretofore, there have been reported as for the fixation of functional substances onto or into a liposome, for example, a method in which fragments of an antibody are bound to an aminoethyl carbamoyl-methyl group substituted on the polysaccharide on the surface of a liposome covered with a pullulan derivative via .gamma.-maleimidobutyloxysuccinimidyl [See "Biochem. Biophys. Acta.", 898, 323 (1987)] and a method in which an antibody is fixed onto a liposome in such a manner that a glycolipid is added preliminarily to the ingredients for forming the liposome membrane and, after the liposome has been formed, a periodate oxidation is carried out and the thereby formed aldehyde group is reacted with the antibody [See "J. Biol. Chem.", 255, 10509 (1980)].
These prior arts include, however, a problem that a multistep chemical reaction on the liposome membrane has to be incorporated after the liposome has been formed and, thus, the amount of the contemplated functional substance introduced is limited to a lower value, with simultaneous high possibility of contamination by the by-products and impurities, bringing about a large probability of damage of the liposome membrane.
On the other hand, it has been pointed out that no sufficient effect is achieved by the use of liposome, since a large part of the liposome is caught by organs in the reticuloendothelial system, such as liver, spleen etc., upon administration of the liposome product [Cancer Res., 43, 5328 (1983)]. In order to solve problems by the above-mentioned liposome capture in the organs of reticuloendothelial system and by the low stability of liposome itself, such as the tendency to collapse and coagulation, attempt has been made to introduce polyethylene glycol chains into the surface layer of liposome [See, for example, WO 90/4384, Japanese Patent Application Kokai No. 249717/1989 and FEBS Letters, 268, 235 (1990)]. Also, it has been made clear that a liposome modified by polyethylene glycol can afford to maintain the liposome concentration in blood for long period of time [Biochem. Biophys. Acta, 1066, 29-36 (1991)].
However, the liposome having introduced therein polyethylene glycol chains does not react with functional substances, so that these functional substances can not be fixed on the liposome surface.
In European Patent Publication No. 526700, it is taught that an antibody-bound liposome containing a drug in which the problem of drug capturing in organs of reticuloendothelial system is improved can be obtained by reacting a maleimide group-containing liposome first with a protein provided with thiol groups (thiolated protein) and reacting, then, the remaining maleimide groups with a compound having a moiety of a polyalkylene glycol having thiol groups (thiolated polyalkylene glycol).
This liposome has, however, a defect that the expected effect is not attained sufficiently, since the antibody is hidden behind the polyalkylene glycol layer and the reaction of the antibody with the target site is obstructed.
In WO 91/16040, a liposome preparation is disclosed, which comprises an anionic group-containing polyethylene glycol derivative, such as an .alpha.-stearyl-.omega.-propionic acid-polyoxyethylene. However, this polyoxyethylene derivative tends to separate off easily from the liposome membrane, since the hydrophobic moiety thereof consists of a monoalkyl group, so that a liposome containing such polyoxyethylene derivative as the membrane-forming component is inferior in the long term stability.